This invention relates to the introduction of diagnostic or therapeutic agents into platelets and more specifically to the use of platelets having associated diagnostic or therapeutic agents for in vivo imaging and targeting.
Platelets are small cells derived from precursor megakaryocytes which control blood clotting and aid in repair of blood vessels. Because of their biological functions, platelets localize to specific areas of diseases including sites of inflammation, infection, thrombosis and neoplastic tumors.
Removal of foreign materials from the blood may also be a major physiological role for platelets. Platelets are known to endocytose a variety of particles in vitro, Clawson, C. C., Am. J. Pathol. 70:449-464, 1973, which is incorporated herein by reference. Particles and solutes may be endocytosed by different mechanisms, Zucker-Franklin, D. J., Cell Biol. 91:706-715, 1981, which is incorporated herein by reference. For example, uptake of particles is energy dependent and arrested when both oxidative phosphorylation and glycolysis are inhibited. Particle uptake is also divalent cation dependent and the process is usually associated with release of platelet granule contents into the extracellular media. On the other hand, the uptake of soluble substances is not energy dependent, is arrested at 4.degree. C. and does not result in degranulation.
Previously, latex spheres have been used as a model for foreign particle ingestion by platelets. Progressive accumulation of latex spheres in the open channel system (OCS), a system of channels believed to be formed by invagination of the plasma membrane, was followed by localization of the particles in acid phosphatase-positive electron-opaque vacuoles, Lewis et al., Blood 47:833-840, 1976, which is incorporated herein by reference. Platelets have also been shown to take up virus particles. Influenza virus, a myxovirus, is taken up by platelets via the OCS, Jerushalmy et al., Proc. Soc. Exp. Biol. Med. 106:462-466, 1962, which is incorporated herein by reference. The mechanism of incorporation and the fate of the virus have not been determined.
Cellular uptake mechanisms such as phagocytosis and viral mediated uptake have been used for introducing drugs and macromolecules in vitro into specific cell types. Such drugs and macromolecules include inorganic and organic compounds, liposomes, red blood cell ghosts and polypeptides. For example, phagocytosis has been used to introduce liposome encapsulated muramyl dipeptide (MDP) into macrophages, Fidler et al., Cancer Res. 49:4665-4670 (1989), which is incorporated herein by reference. Monocytes have also been shown to ingest similar materials in vitro. Viral mediated mechanisms include uptake of Sendai virus, a murine paramyoxovirus, which is capable of entering a cell through membrane fusion.
Although the applications are limited, preferential delivery of drugs and macromolecules to specific cell types in vivo has ben accomplished through nonspecific mechanisms such as phagocytosis. For example, liposomes loaded with agents which activate macrophages and thereby kill tumor cells have been used parenterally to treat tumor metastases. Macrophages are localized to various tissues and organs especially those of the reticuloendothelium system (e.g., liver, spleen, lymph nodes and lungs) and after phagocytosis of liposomes containing activating agents show increased rates of phagocytosis and killing of various infectious agents and tumor cells.
The applications described above cannot be employed for the specific targeting of drugs and macromolecules to many desired locations within an organism. Instead, such applications are limited to specific phagocytotic cell types and have been used to enhance a particular function exhibited by cells, such as enhanced tumoricidal rate of macrophages.
In vivo liposome delivery to macrophages and monocytes is also very inefficient. The percentage of liposomes taken up by these cells is small and therefore requires administration of liposomes in a large excess to achieve an effective delivery. Phagocytosis of the liposomes by other cell types is one factor which can account for this low efficiency. Additionally, the lipid composition of the liposome is another factor which influences their uptake.
Systems which employ particles other than liposomes have been used for diagnostic purposes. Platelets have been labeled with radio-isotopes and used for in vivo imaging. These methods involve chemically linking the imaging reagent to the platelet surface and can result in transfer of the label to other cells within the blood stream after injection. Such label transfer has unnecessary deleterious effects on the receiving cells, results in a lower effective amount to the imaged area and can also affect the resolution of the final image.
There exists a need to be able to target a variety of specific disease locations within an organism, organ or graft with effective amounts of diagnostic and therapeutic agents. The present invention satisfies this need and provides related advantages as well.